Effective degradation of plant biomass into monosaccharides, which are useful building blocks for biochemicals or biofuels, requires a variety of hydrolytic and oxidative enzymes. Such enzymes are preferably thermo-tolerant, since the rigidity of the plant cell walls asks for a thermo-assisted pre-treatment as a first step to increase the accessibility of cell wall polysaccharides (1). Commercial enzyme cocktails for plant cell wall degradation mostly comprise enzymes produced by filamentous fungi like Aspergillus, Trichoderma and Talaromyces strains. In addition, the commercially available fungus Myceliophthora thermophila C1 is a good candidate for the production of thermo-tolerant carbohydrate-active enzymes (2). Still, improvement of already existing enzyme cocktails is required for the success of enzyme-driven biorefinery processes.
Secondary plant cell walls are built from a matrix of cellulose fibrils, interacting with hemicelluloses and glued together with a network of phenolic lignin (3-5). The majority of hemicelluloses is composed of xylan, mannan, and xylo-glucan building blocks, having backbones of β-(1→4) linked xylosyl residues, β-(1→4) linked mannosyl or β-(1→4) linked glucosyl residues, respectively (6). Such hemicelluloses are strongly associated with cellulose via hydrogen-bonding, especially the ones with a low amount of substitution or a block-wise distribution of substituents (3, 5). The cellulose-associated hemicelluloses block cellulases to reach their target substrate, which is likely to contribute to the defense of the plant against microbial attack. Also, hemicelluloses inhibit the deconstruction of plant polysaccharides by commercial enzymes (7, 8) in the formation of fermentable monosaccharides. Hence, degradation of these cellulose-associated hemicelluloses is essential to improve cellulose hydrolysis from plant biomass. As xylan is the main component of hemicellulose, enzymes degrading xylan play a major role in hemicellulose degradation.
Xylan degrading and/or modifying enzymes are broadly applied in industry, such as in upgrading of animal food, in improving the quality of baking and brewing products, in bleaching and modification soft and hardwood kraft pulp, in paper recycling, in macerating vegetables and fruits, in clarifying cereal solution and fruit juices, and in the production of bio fuel and/or other chemicals from residues produced by agriculture and forestry. However, in many of the practical applications, the use of enzymes for xylan degradation and/or modification is not straightforward; the enzymes must be active in the temperature and pH conditions of the process in which they are used. For instance, formulation of commercial feed using pelleting, extrusion or expansion, often contains steps involving high temperatures (70-180° C.) and requires enzymes that withstand these conditions. Furthermore, bleaching processes, and even the sequence of the steps used in the bleaching process, vary among different pulp mills and therefore provide for specific requirements to the enzymes used in these processes. Because of the heterogeneity and complex chemical nature of plant xylan and the broad and heterogenic nature of the applications, there is thus a continuous need to find new xylan degrading and/or modifying enzyme activity, preferably active in different temperature and pH conditions.
Thus, it is an object of the present invention to provide for new means and methods for modifying and/or degrading xylans. The invention provides a newly discovered enzyme activity of a class of fungal enzymes, i.e. thermo-tolerant lytic polysaccharide monooxygenases (LPMOs). A LPMO of the invention is an auxiliary activity family 9 (AA9) LPMO, previously known as GH61 enzymes. These enzymes are described to have cellulolytic and cellulolytic enhancing activities (Morgenstern et al., 2014, Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family, Briefings in Functional Genomics, 13 (6): 421-423). The inventors discovered that the LPMO of the invention not only cleaves celluloses but also cleaves xylans, e.g. in cellulose-associated xylan under formation of oxidised xylo- and gluco-oligomers. In addition, the LPMO displays a synergistic effect when acting together with endoglucanases. The invention further provides methods of using the novel LPMO in a variety of application requiring the cleavage of cellulose and/or xylans.